The TOR (target of rapamycin) and RAS/cyclic AMP (cAMP) signaling pathways are the two major pathways controlling cell growth in response to nutrients in yeast. In this study we examine the functional interaction between TOR and the RAS/cAMP pathway. First, activation of the RAS/cAMP signaling pathway confers pronounced resistance to rapamycin. Second, constitutive activation of the RAS/cAMP pathway prevents several rapamycin-induced responses, such as the nuclear translocation of the transcription factor MSN2 and induction of stress genes, the accumulation of glycogen, the induction of autophagy, the down-regulation of ribosome biogenesis (ribosomal protein gene transcription and RNA polymerase I and III activity), and the down-regulation of the glucose transporter HXT1. Third, many of these TOR-mediated responses are independent of the previously described TOR effectors TAP42 and the type 2A-related protein phosphatase SIT4. Conversely, TOR-controlled TAP42/SIT4-dependent events are not affected by the RAS/cAMP pathway. Finally, and importantly, TOR controls the subcellular localization of both the protein kinase A catalytic subunit TPK1 and the RAS/cAMP signaling-related kinase YAK1. Our findings suggest that TOR signals through the RAS/cAMP pathway, independently of TAP42/SIT4. Therefore, the RAS/cAMP pathway may be a novel TOR effector branch.

In this report, we establish a straightforward method for estimating the equilibrium constant for the creatine kinase reaction (CK K_eq″) over wide but physiologically and experimentally relevant ranges of pH, Mg²⁺ and temperature. Our empirical formula for CK K_eq″ is based on experimental measurements. It can be used to estimate [ADP] when [ADP] is below the resolution of experimental measurements, a typical situation because [ADP] is on the order of micromolar concentrations in living cells and may be much lower in many in vitro experiments. Accurate prediction of [ADP] is essential for in vivo studies of cellular energetics and metabolism and for in vitro studies of ATP-dependent enzyme function under near-physiological conditions. With [ADP], we were able to obtain improved estimates of ΔG_ATP, necessitating the reinvestigation of previously reported ADP- and ΔG_ATP-dependent processes. Application to actomyosin force generation in muscle provides support for the hypothesis that, when [Pi] varies and pH is not altered, the maximum Ca²⁺-activated isometric force depends on ΔG_ATP in both living and permeabilized muscle preparations. Further analysis of the pH studies introduces a novel hypothesis around the role of submicromolar ADP in force generation.

A genomic DNA fragment containing the Raf-1 promoter region was isolated by using a cDNA extension clone. Nucleotide sequencing of genomic DNA clones, primer extension, and S1 nuclease assays have been used to identify the 5' ends of Raf-1 RNAs. Consistent with its ubiquitous expression, the Raf-1 promoter region had features of a housekeeping gene in that it was GC-rich (HTF-like), lacked TATA and CAAT boxes, and contained heterogeneous RNA start sites and four potential binding sites for the transcription factor SP1. In addition, an octamer motif (ATTTCAT), a potential binding site for the octamer family of transcription factors, was located at -734 base pairs. The Raf-1 promoter region drove reporter gene expression 30-fold over the promoterless reporter in Cos 7 cells.

In this report, we establish a straightforward method for estimating the equilibrium constant for the CK reaction over wide but physiologically and experimentally relevant ranges of pH, Mg2+ and temperature. Our empirical formula for CK Keq” is based on experimental measurements. It can be used to estimate [ADP] when [ADP] is below the resolution of experimental measurements, a typical situation because [ADP] is on the order of micromolar concentrations in living cells, and may be much lower in many in vitro experiments. Accurate prediction of [ADP] is essential for in vivo studies of cellular energetics and metabolism, and for in vitro studies of ATP-dependent enzyme function under near-physiological conditions. With [ADP], we could estimate ΔGATP. Application to actomyosin force generation in muscle provides support for the hypothesis that, when [Pi] varies but not when pH is altered, maximum Ca2+-activated isometric force depends on ΔGATP in both living and permeabilized muscle preparations. Further analysis of the pH studies introduces a novel hypothesis for the role of submicromolar ADP in force generation.